Twelve isoforms have been identified in the PKC family of serine/threonine protein kinases, which are encoded by different genes. The PKC isoforms are classified as a result of enzymatic and molecular analysis into three major classes. It is known that the isoforms of PKC are major intracellular mediators that control cell proliferation and differentiation in a variety of cell systems. The expression and function of the isoforms appear to be tissue specific, and the various isoforms appear to be involved in specific physiological processes. In primary human osteoblasts, the role and expression of PKC isoforms remains to be investigated. We hypothesize that the proliferative process in human osteoblast involves specific isoforms of PKC. The aim of this proposal is to investigate PKC isoforms that are expressed by human osteoblasts and to examine the role that specific isoforms play in osteoblastic cell proliferation. Specific aims are: 1) to determine which PKC isoforms is involved in the proliferative process of human osteoblastic cells. First, known activators of human osteoblastic cell proliferation will be used as tools to determine the isoforms involved in proliferation (PDGF, S1P, serum enriched vs serum deprived human osteoblastic cell cultures will be used). The proliferative responses will be monitored by [3H]-thymidine incorporation and the effects on PKC isoform expression and activation will be determined using Western blotting. Second, to better define the role of specific isoforms in proliferation, antisense oligonucleotides to individual PKC isoforms will be used to inhibit the expression of the isoforms and proliferation will be monitored. 2) To investigate the mechanism by which specific PKC isoforms affect osteoblastic cell proliferation. It will be determined if the effects of PKC isoforms are mediated through Gi and MAP kinase dependent pathways by making use of PTX. The information obtained through these studies should elucidate factors regulating proliferation of human osteoblast and aid in the development of rationale therapeutic to control systemic and local bone loss as in osteoporosis and periodontal disease.